JP7697941B2 - Threshold-Based IDA Exclusion List - Google Patents

Description

CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Patent Application No. 62/916,759, filed October 17, 2019, the contents of which are incorporated herein by reference in their entirety.

The teachings herein relate to a mass spectrometer for detecting metabolites of a sample in an Information Dependent Acquisition (IDA) mass spectrometry experiment. More specifically, a mass spectrometer is operated to distinguish metabolite ions from background ions having similar mass/charge ratios (m/z) by including intensity and retention time parameters in an IDA exclusion list.

The apparatus and methods disclosed herein may also be implemented in conjunction with a processor, controller, microcontroller, or computer system, such as the computer system of FIG.
Mass spectrometry background technology

Mass spectrometry (MS) is an analytical technique for the detection and quantification of chemical compounds based on the analysis of the m/z values of ions formed from those compounds. MS involves the ionization of one or more compounds of interest from a sample to produce precursor ions, and mass analysis of the precursor ions.

Tandem mass spectrometry or mass spectrometry/mass spectrometry (MS/MS) involves ionization of one or more compounds of interest from a sample, selection of one or more precursor ions of the one or more compounds, fragmentation of the one or more precursor ions into product ions, and mass analysis of the product ions.

Mass spectrometers are often coupled to chromatography or other separation systems to identify and characterize compounds of interest eluting from a sample. In such coupled systems, compounds in the eluting solvent are ionized and a series of mass spectra are acquired at defined time intervals. These times can range, for example, from 1 second to 100 minutes or more. The intensity values derived from the series of mass spectra form a chromatogram. For example, the sum of all the intensities generates a total ion chromatogram (TIC) and the intensity of one mass value generates an extracted ion chromatogram (XIC).

The peaks found in the chromatogram are used to identify or characterize known peptides or compounds in the sample because they elute at known times, called retention times. More specifically, the retention times of the peaks and/or the areas of the peaks are used to identify or characterize (quantify) known peptides or compounds in the sample.

In a conventional separation-coupled mass spectrometry system, a precursor ion of a known compound is selected for analysis. An MS/MS scan is then performed at each separation interval across a mass range that includes the precursor ion. The intensities of the product ions found in each MS/MS scan are collected over time and analyzed as a collection of spectra, or, for example, XICs.

Both MS and MS/MS can provide qualitative and quantitative information. The measured precursor or product ion spectra can be used to identify the molecule of interest. The intensities of the precursor and product ions can also be used to quantify the amount of the compound present in the sample.

Many different types of experimental acquisition methods or workflows can be performed using tandem mass spectrometers. Three broad categories of these workflows are targeted acquisition, information-dependent acquisition (IDA) or data-dependent acquisition (DDA), and data-independent acquisition (DIA).

In targeted acquisition methods, one or more transitions of precursor ions to product ions are predefined or known for a compound of interest. As a sample is introduced into the tandem mass spectrometer, one or more transitions are monitored during each of a plurality of periods or cycles. In other words, the mass spectrometer selects and fragments the precursor ion of each transition and performs targeted mass analysis on the product ions of the transition. As a result, an intensity (product ion intensity) is generated for each transition. Targeted acquisition methods include, but are not limited to, multiple reaction monitoring (MRM) and selected reaction monitoring (SRM).

In the IDA method, the user can define criteria for performing untargeted mass analysis of the product ions while the sample is being introduced into the tandem mass spectrometer. For example, in the IDA method, a precursor ion or mass spectrometry (MS) survey scan is performed to generate a precursor ion peak list. The user can select criteria for filtering the peak list for a subset of the precursor ions on the peak list. MS/MS is then performed on each precursor ion of the subset of precursor ions. A product ion spectrum is generated for each precursor ion. The MS survey scan followed by multiple MS/MS scans can be repeatedly (iteratively) performed on the precursor ions of the subset of precursor ions as the sample is being introduced into the tandem mass spectrometer. IDA can also be referred to as data-dependent analysis (Thermo Fisher) or data-directed analysis (Waters). For example, the term "DATA-DEPENDENT" is a trademark of Thermo Fisher, and the term "DDA" is a trademark of Waters.

Measurement of complex (e.g. biological) samples by different omics techniques such as proteomics, metabolomics, etc. leads to compounds of different types, large numbers, and wide dynamic ranges. In proteomics and many other sample types, the complexity and dynamic range of compounds is very large. This poses challenges for traditional targeting and IDA methods and demands ultrafast MS/MS acquisition to thoroughly interrogate the sample to both identify and quantify a wide range of analytes.

As a result, a third broad category of tandem mass spectrometry, DIA methods, has been developed. These DIA methods have been used to increase the reproducibility and comprehensiveness of data collection from complex samples. DIA methods can also be called non-specific fragmentation methods. In traditional DIA methods, the operation of the tandem mass spectrometer is not varied between MS/MS scans based on data obtained in previous precursor or product ion scans. Instead, a precursor ion mass range is selected. The precursor ion mass selection window is then stepped across the precursor ion mass range. All precursor ions within the precursor ion mass selection window are fragmented, and all product ions of all precursor ions within the precursor ion mass selection window are mass analyzed.
The problem of endogenous background peaks

When conducting drug metabolite identification studies, IDA is typically performed on samples from medicated subjects. The resulting MS/MS spectra are used to confirm that the putative metabolites are indeed related to the starting drug and to localize the sites of metabolic transformation.

Figure 2 is an exemplary diagram 200 showing how a tandem mass spectrometer operated to identify drug metabolites in a simple sample using an IDA method can generate an IDA list that includes endogenous background precursor ions in addition to metabolite precursor ions. In the IDA method of Figure 2, the tandem mass spectrometer first performs a full MS scan 210 of the simple sample. In the MS scan 210, all precursor ions of the sample are selected in a mass filter, transmitted through a dissociation device, and mass analyzed in a mass analyzer. In this figure, these devices are shown as quadrupoles, but could also be other types of devices. The mass analyzer generates intensity measurements for the precursor ions, as shown in precursor mass spectrum 212.

The tandem mass spectrometer then selects all precursor ions with intensities above a certain threshold 213. All precursor ions with intensities above the threshold 213 are added to a threshold list 214. When the number of ions on the threshold list 214 is small enough to be analyzed by MS/MS within a defined cycle time as shown in FIG. 2, the threshold list 214 becomes an IDA list 216. Note that only the IDA list 214 contains the m/z of each precursor ion. Also note that precursor ions with intensities above the threshold 213 are added to the threshold list 214 according to their m/z values. However, there are other methods for selecting the order of precursor ions on the threshold list 214, as discussed below.

For each precursor ion on the IDA list 216, the tandem mass spectrometer performs an MS/MS scan. For example, in MS/MS scan 221, a precursor ion with m/z value of 114 is selected in the mass filter, dissociated into product ions in the dissociation device, and the product ions are mass analyzed in the mass analyzer. Similarly, in MS/MS scans 222 and 223, precursor ions with m/z values of 153 and 215 from the IDA list 216 are selected and dissociated, respectively, and their product ions are mass analyzed.

The product ion spectra of MS/MS scans 221, 222, and 223 are compared to known product ion spectra for known drugs to determine whether they likely correspond to metabolites. Note that the precursor ions with m/z values of 114 and 153 are endogenous background ion peaks. As a result, the endogenous background ion peaks can also be compared to known metabolite peaks in the IDA method. For simple samples, this is not an issue.

However, for more complex samples, this can prevent the IDA method from triggering on one or all of the metabolite peaks because much of the available cycle time is used to obtain unwanted MS/MS spectra of endogenous background peaks. As a result, drug metabolites may not be identified.

Figure 3 is an exemplary diagram 300 showing how a tandem mass spectrometer operated to identify drug metabolites in a complex sample using an IDA method can generate an IDA list that does not include metabolite precursor ions. In the IDA method of Figure 3, the tandem mass spectrometer first performs a full MS scan 310 of the complex sample. In the MS scan 310, all precursor ions of the sample are selected in a mass filter, transmitted through a dissociation device, and mass analyzed in a mass analyzer. In this figure, these devices are shown as quadrupoles, but could also be other types of devices. The mass analyzer generates intensity measurements for the precursor ions, as shown in precursor mass spectrum 312.

The tandem mass spectrometer then selects all precursor ions with intensities above a certain threshold 313. All precursor ions with intensities above the threshold 313 are added to a threshold list 314.

Because a complex sample is scanned, the precursor mass spectrum 312 contains, in addition to the metabolite peaks, six endogenous background precursor ion peaks, which have m/z values of 215. If the defined cycle time provides enough time for an MS/MS scan to be performed on only five precursor ion peaks, the threshold list 314 is compiled into an IDA list 316.

For each precursor ion on the IDA list 316, the tandem mass spectrometer performs an MS/MS scan. Because there are five precursor ion peaks on the IDA list 316, five MS/MS scans 321-325 are performed to generate five product ion spectra.

The product ion spectra of MS/MS scans 321-325 are then compared to known product ion spectra for known drugs to determine whether they likely correspond to metabolites. Metabolites may not be "known" per se. Note that in precursor ion spectrum 312, the metabolite precursor ion with m/z 215 is preceded by six endogenous background precursor ions. Because the IDA list 316 is limited to only five precursor ion peaks, the metabolite precursor ion with m/z 215 was not added to the list. As a result, the product ion spectra of MS/MS scans 321-325 provide only product ions of the endogenous background precursor ions. As a result, no known drug metabolites are found in this case.

Performing analysis via IDA acquisition is one of the most widely used methods of generating MS/MS information in an automated fashion. Over the years, several filters for filtering threshold peak lists for a subset of precursor ions to be dissociated have been developed to optimize the automated selection of ions of interest in specific applications.

In a metabolism study, a control sample can be obtained and analyzed. The control sample is similar to the metabolism sample, but represents the condition prior to administration of the drug. Naturally, the more similar the control sample is to the experimental sample, the better. For example, the control sample can be pre-dose, but from the same subject as the corresponding medicated sample. Putative metabolites are found by looking for peaks that appear in the medicated sample but not in the control sample.

By finding all peaks prior to acquisition of the medicated sample, it is possible to analyze the control sample. It has been shown previously that this list of peaks can be used as an IDA exclusion list when acquiring data on the medicated sample. This prevents the IDA from being triggered on background peaks, thus increasing the likelihood that MS/MS will be acquired on the actual drug metabolite of interest.

Figure 4 is an exemplary diagram 400 showing how a tandem mass spectrometer can be operated to first perform an MS scan on a control sample to generate an exclusion list, and then to identify drug metabolites in a complex sample by using the exclusion list in an IDA method applied to the complex sample. In the IDA method of Figure 4, the tandem mass spectrometer first performs a full MS scan 401 of the control sample. The control sample is known to not contain any metabolites of interest. In the MS scan 401, all precursor ions of the control sample are selected in a mass filter, transmitted through a dissociation device, and mass analyzed in a mass analyzer. In this figure, these devices are shown as quadrupoles, but could also be other types of devices. The mass analyzer generates intensity measurements for the precursor ions, as shown in the precursor mass spectrum 402.

The tandem mass spectrometer then selects all precursor ions with intensities above a certain threshold 403. All precursor ions with intensities above the threshold 403 are added to an exclusion list 404. Because the control sample does not contain the metabolite of interest, all precursor ions on the exclusion list 404 are endogenous background ions.

The tandem mass spectrometer then performs a full MS scan 410 of the experimental sample known to contain the metabolite of interest. In the MS scan 410, all precursor ions of the experimental sample are selected in a mass filter, transmitted through a dissociation device, and mass analyzed in a mass analyzer. In this diagram, these devices are shown as quadrupoles, but could also be other types of devices. The mass analyzer produces intensity measurements for the precursor ions, as shown in precursor mass spectrum 412.

The tandem mass spectrometer then selects all precursor ions with intensities above a certain threshold 413. All precursor ions with intensities above the threshold 413 are added to a threshold list 414. Because the experimental sample contains a metabolite of interest, the threshold list 414 contains the metabolite precursor ion with m/z 215 in addition to all endogenous background ions.

The tandem mass spectrometer removes precursor ion peaks that are on the exclusion list 404 from the threshold list 414. This generates an IDA list 416, which now contains only metabolite precursor ions with m/z 215.

For each precursor ion on the IDA list 416, the tandem mass spectrometer performs an MS/MS scan. Because there is only one precursor ion peak on the IDA list 416, only an MS/MS scan 421 is performed to produce one product ion spectrum.

The product ion spectra of the MS/MS scan 421 are then compared to known product ion spectra for known drugs to determine whether they likely correspond to metabolites. Note that by using the exclusion list 404, the MS/MS scan is not performed on endogenous background precursor ion peaks. This ensures that the MS/MS scan 421 is performed on the metabolite precursor ion with m/z 215.

One problem with using an exclusion list derived from a control sample is that MS/MS will not trigger for peaks for drug metabolites that have m/z similar to background peaks, even if the peak intensity is significantly greater for the drug metabolite compared to the background peak.

Figure 5 is an exemplary diagram 500 showing how an exclusion list from an MS scan performed on a control sample may also remove metabolite peaks in an IDA method if the metabolite peak and background peaks have similar m/z values. In the IDA method of Figure 5, the tandem mass spectrometer first performs a full MS scan 501 of the control sample. The control sample is known to be free of metabolites of interest. In the MS scan 501, all precursor ions of the control sample are selected in a mass filter, transmitted through a dissociation device, and mass analyzed in a mass analyzer. In this figure, these devices are shown as quadrupoles, but could also be other types of devices. The mass analyzer produces intensity measurements for the precursor ions, as shown in precursor mass spectrum 502.

The tandem mass spectrometer then selects all precursor ions with intensities above a certain threshold 503. All precursor ions with intensities above the threshold 503 are added to an exclusion list 504.

The tandem mass spectrometer then performs a full MS scan 510 of the experimental sample known to contain the metabolite of interest. In the MS scan 510, all precursor ions of the experimental sample are selected in a mass filter, transmitted through a dissociation device, and mass analyzed in a mass analyzer. In this illustration, these devices are shown as quadrupoles, but could also be other types of devices. The mass analyzer produces intensity measurements for the precursor ions, as shown in precursor mass spectrum 512.

The tandem mass spectrometer then selects all precursor ions with intensities above a certain threshold 513. All precursor ions with intensities above the threshold 513 are added to a threshold list 514. Because the experimental sample contains a metabolite of interest, the threshold list 514 contains a metabolite precursor ion 518 with m/z 190. However, a background ion 517 with m/z 190 is also present. As a result, the threshold list 514 contains two ion peaks with m/z 190.

The tandem mass spectrometer then removes the precursor ion peaks that are on the exclusion list 504 from the threshold list 514. Because metabolite precursor ion 518 and background ion 517 have the same m/z value of 190, they are both excluded. As a result, the IDA list 516 now does not contain any ion peaks and no MS/MS scan is performed. As a result, when metabolite and background peaks have similar m/z values, no MS/MS scan is triggered for the drug metabolite even when control samples and exclusion lists are used.

As a result, additional system methods for operating a tandem mass spectrometer in an IDA method are required to distinguish between background and metabolite ions with similar m/z values.

A system, method, and computer program product are disclosed for removing endogenous background ions having the same mass/charge ratio (m/z) as metabolite ions from a peak list in an information-dependent acquisition (IDA) mass spectrometry experiment. The system includes an ion source device and a tandem mass spectrometer.

The tandem mass spectrometer receives an ion beam from the ion source device that is generated by ionizing a control sample that does not contain metabolite compounds. The tandem mass spectrometer performs an MS scan of a mass range on the ion beam to generate background peak m/z and intensity values for the background precursor ions. The tandem mass spectrometer selects one or more of the background peaks of the background precursor ions for an exclusion list and includes the m/z and intensity values for each background peak of the selected one or more background peaks in the exclusion list.

The tandem mass spectrometer then creates a peak list for the IDA method by analyzing the experimental sample. The tandem mass spectrometer receives an ion beam from an ion source device that is generated by ionizing an experimental sample containing metabolite compounds. The tandem mass spectrometer performs an MS scan of the mass range of the ion beam to generate peak m/z and intensity values for the precursor ions. The tandem mass spectrometer selects one or more of the peaks of the precursor ions for the peak list and includes the per-peak m/z and intensity values of the selected one or more peaks in the peak list.

Finally, the tandem mass spectrometer excludes from the peak list each peak that has both an m/z value and an intensity value that correspond to the m/z value and intensity value of a background peak in the exclusion list.

These and other features of the applicants' teachings are described herein.
The present specification also provides, for example, the following items:
(Item 1)
1. A system for removing endogenous background ions having the same mass/charge ratio (m/z) as a metabolite ion from a peak list in an Information Dependent Acquisition (IDA) mass spectrometry experiment, comprising:
an ion source device;
A tandem mass spectrometer, comprising:
(a) generating an exclusion list by receiving an ion beam from the ion source device generated by ionizing a control sample that does not contain metabolite compounds, performing a mass spectrometry (MS) scan of a mass range on the ion beam to generate background peak m/z and intensity values for background precursor ions, selecting one or more of the background peaks of the background precursor ions for an exclusion list, and including the m/z value and intensity value for each background peak of the selected one or more background peaks in the exclusion list;
(b) generating said peak list by receiving an ion beam from said ion source device generated by ionizing an experimental sample containing said metabolite compounds, performing an MS scan of said ion beam of said mass range to generate peak m/z and intensity values for precursor ions, selecting one or more of said precursor ion peaks for a peak list, and including per-peak m/z and intensity values of said selected one or more peaks in said peak list;
(c) excluding from the peak list each peak having both an m/z value and an intensity value that corresponds to an m/z value and an intensity value of a background peak in the exclusion list;
A tandem mass spectrometer and
A system comprising:
(Item 2)
2. The system of claim 1, wherein the m/z and intensity values of a peak in the peak list correspond to the m/z and intensity values of a background peak in the exclusion list if the m/z value fits within an m/z tolerance factor and the intensity value fits within an intensity tolerance factor.
(Item 3)
2. The system of claim 1, wherein the tandem mass spectrometer further scales the peak list or the exclusion list before comparing the peaks in step (c).
(Item 4)
The tandem mass spectrometer further comprises:
selecting a known background peak that is on the peak list and the exclusion list;
calculating a ratio of an intensity value of the one known background peak on the peak list and an intensity value of the one known background peak on the exclusion list;
multiplying each intensity value on the peak list or the exclusion list by the ratio;
5. The system of claim 4, wherein the intensities of the peak list or the exclusion list are scaled by:
(Item 5)
The tandem mass spectrometer further comprises:
selecting two or more known background peaks that are on the peak list and the exclusion list;
calculating a ratio of a combination of intensity values of the two or more known background peaks on the peak list and a combination of intensity values of the two or more known background peaks on the exclusion list;
multiplying each intensity value on the peak list or the exclusion list by the ratio;
5. The system of claim 4, wherein the intensities of the peak list or the exclusion list are scaled by:
(Item 6)
The tandem mass spectrometer further includes a sample introduction device, the tandem mass spectrometer further comprising:
In step (a), receiving an ion beam from the ion source device receiving one or more compounds from the control sample over time from the sample introduction device, performing a plurality of MS scans of the mass range on the ion beam at a plurality of different time steps to generate background peak m/z and intensity values for background precursor ions over time, and at each time step selecting one or more of the background peaks of the background precursor ions for the exclusion list, and including in the exclusion list the m/z value, intensity value, and residence time value for each background peak of the selected one or more of the background peaks;
In step (b), receiving an ion beam from the ion source device receiving one or more compounds from the experimental sample over time from the sample introduction device, performing a plurality of MS scans of the mass range on the ion beam at a plurality of different time steps to generate peak m/z and intensity values for precursor ions over time, and at each time step selecting one or more of the precursor ion peaks for the peak list, and including per-peak m/z values, intensity values, and dwell time values for the selected one or more peaks in the peak list;
In step (c), excluding from the peak list each peak having an m/z value, intensity value, and retention time corresponding to an m/z value, intensity value, and retention time of a background peak in the exclusion list;
2. The system according to item 1.
(Item 7)
2. The system of claim 1, wherein the m/z values, intensity values, and retention times of peaks in the peak list correspond to the m/z values, intensity values, and retention times of background peaks in the exclusion list if the m/z values fit within an m/z tolerance factor, if the intensity values fit within an intensity tolerance factor, and if the retention time values fit within a retention time tolerance factor.
(Item 8)
1. A method for removing endogenous background ions having the same mass/charge ratio (m/z) as a metabolite ion from a peak list in an Information Dependent Acquisition (IDA) mass spectrometry experiment, comprising:
receiving an ion beam from an ion source device generated by ionizing a control sample that does not contain metabolite compounds using a tandem mass spectrometer;
performing a mass spectrometry (MS) scan of a mass range on the ion beam using the tandem mass spectrometer to generate background peak m/z and intensity values for background precursor ions;
using the tandem mass spectrometer to select one or more of the background peaks of the background precursor ions for an exclusion list and include an m/z value and an intensity value for each background peak of the selected one or more background peaks in the exclusion list;
using the tandem mass spectrometer to receive an ion beam from the ion source device that is generated by ionizing an experimental sample that includes the metabolite compounds;
performing an MS scan of the mass range on the ion beam using the tandem mass spectrometer to generate peak m/z and intensity values for precursor ions;
using the tandem mass spectrometer to select one or more of the precursor ion peaks for a peak list and include per-peak m/z and intensity values of the selected one or more peaks in the peak list;
using the tandem mass spectrometer to exclude from the peak list each peak having both an m/z value and an intensity value that corresponds to an m/z value and an intensity value of a background peak in the exclusion list;
A method comprising:
(Item 9)
9. The method of claim 8, wherein the m/z and intensity values of the peaks in the peak list correspond to the m/z and intensity values of the background peaks in the exclusion list if the m/z values fit within an m/z tolerance factor and if the intensity values fit within an intensity tolerance factor.
(Item 10)
9. The method of claim 8, further comprising the step of scaling the peak list or the exclusion list before excluding from the peak list each peak having both an m/z value and an intensity value corresponding to an m/z value and an intensity value of a background peak in the exclusion list.
(Item 11)
Scaling the peak list or the exclusion list comprises:
selecting a known background peak that is on the peak list and the exclusion list;
calculating a ratio of an intensity value of the one known background peak on the peak list and an intensity value of the one known background peak on the exclusion list;
multiplying each intensity value on the peak list or the exclusion list by the ratio;
11. The method according to claim 10, comprising:
(Item 12)
Scaling the peak list or the exclusion list comprises:
selecting two or more known background peaks that are on the peak list and the exclusion list;
calculating a ratio of a combination of intensity values of the two or more known background peaks on the peak list and a combination of intensity values of the two or more known background peaks on the exclusion list;
multiplying each intensity value on the peak list or the exclusion list by the ratio;
11. The method according to claim 10, comprising:
(Item 13)
receiving an ion beam from the ion source device that receives one or more compounds from the control sample over time from a sample introduction device;
performing a plurality of MS scans of the mass range on the ion beam at a plurality of different time steps to generate background peak m/z and intensity values for background precursor ions over time;
At each time step, selecting one or more of the background peaks of the background precursor ions for the exclusion list and including in the exclusion list an m/z value, an intensity value, and a retention time value for each background peak of the selected one or more of the background peaks;
receiving an ion beam from the ion source device that receives one or more compounds from the experimental sample over time from the sample introduction device;
performing a plurality of MS scans of the mass range on the ion beam at a plurality of different time steps to generate peak m/z and intensity values for precursor ions over time;
At each time step, selecting one or more of the precursor ion peaks for the peak list and including per-peak m/z, intensity and retention time values of the selected one or more peaks in the peak list;
excluding from the peak list each peak having an m/z value, an intensity value, and a retention time value corresponding to an m/z value, an intensity value, and a retention time value of a background peak in the exclusion list;
9. The method of claim 8, further comprising:
(Item 14)
14. The method of claim 13, wherein the m/z values, intensity values, and dwell times of the peaks in the peak list correspond to the m/z values, intensity values, and dwell times of the background peaks in the exclusion list if the m/z values fit within an m/z tolerance factor, if the intensity values fit within an intensity tolerance factor, and if the dwell time values fit within a dwell time tolerance factor.
(Item 15)
1. A computer program product comprising a non-transitory, tangible computer readable storage medium, the contents of which include a program with instructions for execution on a processor to perform a method for removing endogenous background ions having the same mass/charge ratio (m/z) as metabolite ions from a peak list in an Information Dependent Acquisition (IDA) mass spectrometry experiment, the method comprising:
Providing a system, the system comprising one or more distinct software modules, the distinct software modules comprising a control module and an analysis module;
using the control module to instruct the tandem mass spectrometer to receive an ion beam from an ion source device that is generated by ionizing a control sample that does not contain metabolite compounds;
using the control module to instruct the tandem mass spectrometer to perform a mass spectrometry (MS) scan of a mass range on the ion beam to generate background peak m/z and intensity values for background precursor ions;
using the analysis module to select one or more of the background peaks of the background precursor ions for an exclusion list and include in the exclusion list an m/z value and an intensity value for each background peak of the selected one or more background peaks;
using the control module to instruct the tandem mass spectrometer to receive an ion beam from the ion source device generated by ionizing an experimental sample containing the metabolite compounds;
using the control module to instruct the tandem mass spectrometer to perform an MS scan of the mass range on the ion beam to generate peak m/z and intensity values for precursor ions;
using the analysis module to select one or more of the precursor ion peaks for a peak list and include per-peak m/z and intensity values of the selected one or more peaks in the peak list;
using the analysis module to exclude from the peak list each peak having both an m/z value and an intensity value that corresponds to an m/z value and an intensity value of a background peak in the exclusion list;
2. A computer program product comprising:

Those skilled in the art will appreciate that the drawings described below are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

FIG. 1 is a block diagram illustrating a computer system upon which embodiments of the present teachings may be implemented.

FIG. 2 is an exemplary schematic diagram showing how a tandem mass spectrometer operated to identify drug metabolites in a simple sample using an information-dependent acquisition (IDA) method can generate an IDA list that includes endogenous background precursor ions in addition to metabolite precursor ions.

FIG. 3 is an exemplary schematic diagram showing how a tandem mass spectrometer operated to identify drug metabolites in a complex sample using the IDA method can generate an IDA list that does not include metabolite precursor ions.

FIG. 4 is an exemplary schematic diagram showing how a tandem mass spectrometer can be operated to first perform a mass spectrometry (MS) scan on a control sample and generate an exclusion list, and then to identify drug metabolites in a complex sample by using the exclusion list in an IDA method applied to the complex sample.

FIG. 5 is an exemplary diagram showing how an exclusion list from an MS scan performed on a control sample can also remove metabolite peaks in an IDA method when the metabolite peak and background peak have similar m/z values.

FIG. 6 is an exemplary diagram showing how adding intensity and retention time to an exclusion list from an MS scan performed on a control sample, according to various embodiments, can prevent a metabolite peak from being removed in an IDA method when the metabolite peak and background peak have similar m/z values.

FIG. 7 is a schematic diagram of an apparatus for removing endogenous background ions having the same m/z as metabolite ions from a peak list in an IDA mass spectrometry experiment, according to various embodiments.

FIG. 8 is a flow chart showing a method 800 for removing endogenous background ions with the same m/z as metabolite ions from a peak list in an IDA mass spectrometry experiment, according to various embodiments.

FIG. 9 is a schematic diagram of a system including one or more distinct software modules that implement a method for excluding endogenous background ions having the same m/z as metabolite ions from a peak list in an IDA mass spectrometry experiment, according to various embodiments.

Before one or more embodiments of the present teachings are described in detail, those skilled in the art will understand that the present teachings are not limited in their application to the details of construction, arrangement of components, and arrangement of steps set forth in the following detailed description or illustrated in the drawings. It is also to be understood that the phraseology and terminology used herein are for the purpose of description and should not be regarded as limiting.

Description of Various Embodiments Computer Implemented System

1 is a block diagram illustrating a computer system 100 in which an embodiment of the present teachings may be implemented. The computer system 100 includes a bus 102 or other communication mechanism for communicating information and a processor 104 coupled with the bus 102 for processing information. The computer system 100 also includes a memory 106, which may be a random access memory (RAM) or other dynamic storage device, coupled to the bus 102 for storing instructions executed by the processor 104. The memory 106 may also be used to store temporary variables or other intermediate information during execution of instructions executed by the processor 104. The computer system 100 further includes a read-only memory (ROM) 108 or other static storage device coupled to the bus 102 for storing static information and instructions for the processor 104. A storage device 110, such as a magnetic disk or optical disk, is provided and coupled to the bus 102 for storing information and instructions.

The computer system 100 may be coupled via the bus 102 to a display 112, such as a cathode ray tube (CRT) or liquid crystal display (LCD), for displaying information to a computer user. An input device 114, including alphanumeric and other keys, is coupled to the bus 102 for communicating information and command selections to the processor 104. Another type of user input device is a cursor control 116, such as a mouse, trackball, or cursor direction keys, for communicating directional information and command selections to the processor 104 and for controlling cursor movement on the display 112. This input device typically has two degrees of freedom in two axes, a first axis (i.e., x) and a second axis (i.e., y), that allow the device to specify a position in a plane.

A computer system 100 can implement the present teachings. According to one implementation of the present teachings, results are provided by the computer system 100 in response to the processor 104 executing one or more sequences of one or more instructions contained in the memory 106. Such instructions may be read into the memory 106 from another computer-readable medium, such as the storage device 110. Execution of the sequences of instructions contained in the memory 106 causes the processor 104 to perform the processes described herein. Alternatively, hardwired circuitry may be used in place of or in combination with software instructions to implement the present teachings. Thus, implementation of the present teachings is not limited to any specific combination of hardware circuitry and software.

In various embodiments, computer system 100 can be connected to one or more other computer systems, such as computer system 100, across a network to form a networked system. The network can include a private network or a public network, such as the Internet. In a networked system, one or more computer systems can store and serve data to other computer systems. The one or more computer systems that store and serve data may be referred to as servers or clouds in a cloud computing scenario. The one or more computer systems may include, for example, one or more web servers. The other computer systems that send and receive data to and from the servers or clouds may be referred to as, for example, clients or cloud devices.

The term "computer-readable medium" as used herein refers to any medium that participates in providing instructions to the processor 104 for execution. Such media may take many forms, including but not limited to non-volatile media, volatile media, and transmission media. Non-volatile media include, for example, optical or magnetic disks, such as storage device 110. Volatile media include dynamic memory, such as memory 106. Transmission media include coaxial cables, copper wire, and fiber optics, including the wires that comprise bus 102.

Common forms of computer-readable media or computer program products include, for example, a floppy disk, a flexible disk, a hard disk, a magnetic tape or any other magnetic medium, a CD-ROM, a digital video disk (DVD), a Blu-ray disk, any other optical medium, a thumb drive, a memory card, RAM, a PROM, and an EPROM, a flash-EPROM, any other memory chip or cartridge, or any other tangible medium from which a computer can read.

Various forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to the processor 104 for execution. For example, the instructions may initially be carried on a magnetic disk of a remote computer. The remote computer can load the instructions into its dynamic memory and send the instructions over a telephone line using a modem. A modem local to the computer system 100 can receive the data on the telephone line and use an infrared transmitter to convert the data to an infrared signal. An infrared detector coupled to the bus 102 can receive the data carried in the infrared signal and place the data on the bus 102. The bus 102 carries the data to memory 106, from which the processor 104 reads and executes the instructions. The instructions received by memory 106 may optionally be stored on storage device 110 either before or after execution by the processor 104.

According to various embodiments, instructions configured to be executed by a processor to perform the method are stored on a computer-readable medium. The computer-readable medium can be a device that stores digital information. For example, computer-readable media include compact disc read-only memories (CD-ROMs), as are well known in the art, for storing software. The computer-readable medium is accessed by a suitable processor to execute the instructions configured to be executed.

The following description of various implementations of the present teachings is presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the present teachings to the precise form disclosed. Modifications and variations are possible in light of the above teachings or may be acquired from practice of the present teachings. In addition, while the described implementations include software, the present teachings may be implemented as a combination of hardware and software or in hardware alone. The present teachings may be implemented with both object-oriented and non-object-oriented programming systems.
IDA Exclusion List with Strength and Dwell Time

As explained above, when performing drug metabolite identification studies, IDA is typically performed on samples from medicated subjects. The resulting MS/MS spectra are used to confirm that the putative metabolites are indeed related to the starting drug and to localize the sites of metabolic transformation. For simple samples, conventional IDA works well. However, for more complex samples, endogenous background ions may prevent the IDA method from triggering for one or all of the metabolite peaks because much of the available cycle time is used to obtain unneeded MS/MS spectra of endogenous background peaks. As a result, drug metabolites may not be identified.

One solution to this problem is to first analyze a similar control sample that does not contain the metabolite to generate a background ion peak list. This list can then be used as an IDA exclusion list when obtaining data for dosed samples. This prevents the IDA from being triggered on background peaks, thus increasing the likelihood that MS/MS will be obtained for the actual drug metabolite of interest.

One problem with using an exclusion list derived from a control sample is that MS/MS will not trigger for peaks related to drug metabolites that have m/z values similar to the background peaks. As a result, additional system methods for operating the tandem mass spectrometer in the IDA method are required to distinguish between background and metabolite ions with similar m/z values.

In various embodiments, the tandem mass spectrometer includes the intensity of each background peak in an IDA exclusion list. The IDA method then triggers an MS/MS scan on the dosed sample if either there is no corresponding peak on the control sample or the peak in the control sample is smaller in intensity. A tolerance factor can be used in comparing peak intensities. For example, the IDA method triggers an MS/MS scan only if the intensity is 1.5 times or greater than that of the background peak. This modification of the IDA method reduces the likelihood that an MS/MS scan will not be triggered for a drug metabolite when using an exclusion list.

As explained above, mass spectrometers are often coupled with chromatography or other separation systems to identify and characterize compounds of interest eluting from a sample. In such coupled systems, compounds in the eluting solvent are ionized and a series of mass spectra are acquired at defined time intervals. The time it takes for an analyte or metabolite to elute from injection to detection is referred to as the retention time (RT). As a result, metabolite ions can also be differentiated from endogenous background ions based on their retention time.

In various embodiments, the tandem mass spectrometer thus includes the intensity and retention time of each background peak in an IDA exclusion list. The IDA method then triggers an MS/MS scan on the dosed sample if either there is no corresponding peak on the control sample or the peak in the control sample has the same retention time and is smaller in intensity.

Figure 6 is an exemplary diagram 600 showing how adding intensity and dwell time to an exclusion list from an MS scan performed on a control sample can prevent a metabolite peak from being removed in an IDA method when the metabolite peak and background peak have similar m/z values, according to various embodiments. In the IDA method of Figure 6, the tandem mass spectrometer first performs several full MS scans of the control sample as it is eluted using a separation device. The three-dimensional chromatogram 602 shows the mass peaks generated for each of the four endogenous background ions. The control sample is known to be free of metabolites of interest. In each MS scan 601 performed at different time steps, all precursor ions of the control sample are selected in a mass filter, transmitted through a dissociation device, and mass analyzed in a mass analyzer to generate a precursor ion spectrum for each time step. In this figure, these devices are shown as quadrupoles, but could also be other types of devices.

At each time step, the tandem mass spectrometer then selects all peaks with intensities above a certain threshold 603. All precursor ion peaks with intensities above the threshold 603 are added to an exclusion list 604. In addition, the intensity and residence time of each peak are also added to the exclusion list 604, which now produces three columns in the exclusion list 604.

The tandem mass spectrometer then performs several full MS scans 610 of an experimental sample known to contain the metabolite of interest. In each MS scan 610, performed at a different time step, all precursor ions of the experimental sample are selected in a mass filter, transmitted through a dissociation device, and mass analyzed in a mass analyzer to generate a precursor ion spectrum for each time step. A three-dimensional chromatogram 612 shows the mass peaks generated.

The tandem mass spectrometer then selects all peaks with an intensity above a certain threshold 613. All precursor ion peaks with an intensity above the threshold 613 are added to a threshold list 614. In addition, the intensity and retention time of each peak are also added to the threshold list 614, now generating three columns in the threshold list 614. Because the experimental sample contains a metabolite of interest, the threshold list 614 includes a metabolite precursor ion peak 618 with m/z 190, intensity 1500, and retention time 41 minutes. However, a background ion peak 617 with m/z 190, intensity 1000, and retention time 41 minutes is also present. As a result, the threshold list 614 includes two ion peaks with m/z 190.

The tandem mass spectrometer then removes the precursor ion peaks that are on the exclusion list 604 from the threshold list 614. Previously, metabolite precursor ion peak 618 and background ion peak 617 in threshold list 614 were both excluded because they had the same m/z value of 190 as the background ion peak in exclusion list 604.

However, now, the retention times and intensities of the precursor ion peak 618 and background ion peak 617 in the threshold list 614 are additionally compared to the retention times and intensities of the background ion peaks in the exclusion list 604. From this comparison, the background ion peak 617 in the threshold list 614 is found to match the background ion peak in the exclusion list 604 and is excluded. However, even though the m/z and retention time of the metabolite precursor ion peak 618 in the threshold list 614 matches the m/z and retention time of the background ion peak in the exclusion list 604, the intensity does not match. As a result, the metabolite precursor ion peak 618 in the threshold list 614 is not excluded and is therefore added to the IDA list 616. Note that all other peaks in the threshold list 614 match the peaks in the exclusion list 604 and are excluded.

Also note that although the IDA list 616 is shown as a separate list, the IDA list 616 could simply be the threshold list 614 after peaks have been removed. Furthermore, note that the threshold list 614 is constantly being updated and removed in real time as peaks are acquired during the separation, and thus the IDA list 616 is also constantly changing during the separation.

During separation, for each precursor ion peak on the IDA list 616, the tandem mass spectrometer performs an MS/MS scan while the precursor ion peak is on the IDA list 616. In this case, since there is only one precursor ion peak on the IDA list 616, only an MS/MS scan 621 is performed as long as the metabolite precursor ion peak 618 is on the IDA list 616, producing a product ion spectrum for each MS/MS scan.

The product ion spectra of the MS/MS scan 621 are then compared to known product ion spectra for known drugs to determine whether they likely correspond to metabolites. Note that by using the exclusion list 604 with additional intensity and retention time parameter values, MS/MS scans are not performed on both endogenous background precursor ion peaks and metabolite ion peaks that have similar m/z values but different intensities or different retention times. In other words, metabolite ion peaks are no longer erroneously excluded.

In various embodiments, when comparing m/z, dwell time, and intensity between the threshold list and the exclusion list, different tolerance factors are used for matching values of each parameter. For example, the tolerance factor for intensity can be, but is not limited to, a factor of 1.5. This means that if the m/z and dwell also match, an MS/MS scan will only be triggered for a peak in the threshold list 614 if the intensity is 1.5 times the intensity of the peak on the exclusion list 604.

Note that the concentrations of the control and experimental samples may differ. As a result, in various embodiments, the intensities in the threshold list 614 or the exclusion list 604 are scaled before the two lists are compared. For example, the control and experimental urine samples may have different volumes. Urine is known to naturally contain certain compounds, such as creatinine. As a result, the ratio of the intensities for creatinine in the threshold list and the exclusion list can be used to scale all intensities in the threshold list or the exclusion list.
A system for removing background peaks from a peak list

Figure 7 is a schematic diagram 700 of an apparatus for removing endogenous background ions having the same m/z as metabolite ions from a peak list in an IDA mass spectrometry experiment, according to various embodiments. The system of Figure 7 includes an ion source device 710 and a tandem mass spectrometer 701.

The ion source device 710 may be, but is not limited to, an electrospray ion source (ESI) device, a chemical ionization (CI) source device such as an atmospheric pressure chemical ionization source (APCI) device, an atmospheric pressure photoionization (APPI) source device, or a matrix-assisted laser desorption source (MALDI) device. In an exemplary embodiment, the ion source device 710 is an ESI device.

The tandem mass spectrometer 701 includes, for example, a mass filter device 720, a dissociation device 730, and a mass analyzer 740. In the system of FIG. 7, the mass filter device 720, the dissociation device 730, and the mass analyzer 740 are shown as quadrupole devices. One skilled in the art can understand that any of these stages may include other types of mass analysis devices, including, but not limited to, an ion trap, an orbitrap, an ion mobility device, a time-of-flight (TOF) device, an electron-based dissociation (ExD) collision cell, or a Fourier transform ion cyclotron resonance (FT-ICR) device.

The tandem mass spectrometer 701 first creates an exclusion list by analyzing a control sample. The tandem mass spectrometer 701 receives an ion beam from an ion source device 710 that is generated by ionizing a control sample that does not contain metabolite compounds. Note that in FIG. 7, the ion source device 710 is shown as part of the tandem mass spectrometer 701. However, the ion source device 710 can also be a separate device.

The tandem mass spectrometer 701 performs an MS scan of a mass range on the ion beam to generate background peak m/z and intensity values for the background precursor ions. The tandem mass spectrometer 701 selects one or more of the background peaks of the background precursor ions for an exclusion list and includes the m/z and intensity values for each background peak of the selected one or more background peaks in the exclusion list.

The tandem mass spectrometer 701 then creates a peak list for the IDA method by analyzing the experimental sample. The tandem mass spectrometer 701 receives an ion beam from an ion source device that is generated by ionizing an experimental sample containing metabolite compounds. The tandem mass spectrometer 701 performs an MS scan of the mass range of the ion beam to generate peak m/z and intensity values for precursor ions. The tandem mass spectrometer 701 selects one or more of the peaks of the precursor ions for the peak list and includes the per-peak m/z and intensity values of the selected one or more peaks in the peak list.

Finally, the tandem mass spectrometer 701 excludes each peak in the peak list that has both an m/z value and an intensity value that correspond to the m/z value and intensity value of a background peak in the exclusion list.

In various embodiments, the tandem mass spectrometer 701 further performs an MS/MS scan of each precursor ion peak on the peak list to identify the metabolite compound.

In various embodiments, the tandem mass spectrometer 701 selects one or more of the precursor ion peaks for the peak list by selecting the first N peaks that are measured above a certain threshold intensity level. In another embodiment, the tandem mass spectrometer 701 selects one or more of the precursor ion peaks for the peak list by selecting the N peaks with the highest intensities.

In various embodiments, the m/z and intensity values of a peak in the peak list correspond to the m/z and intensity values of a background peak in the exclusion list if the m/z value fits within the m/z tolerance factor and the intensity value fits within the intensity tolerance factor. As explained above, the intensity tolerance factor can be, for example, 1.5 times the intensity of the background peak in the exclusion list.

In various embodiments, the tandem mass spectrometer 701 further scales the peak list or the exclusion list before comparing and matching the peaks in the two lists. As explained above, a single known background peak can be used to scale the peak list or the exclusion list. For example, the tandem mass spectrometer 701 selects one known background peak that is on the peak list and the exclusion list. The tandem mass spectrometer 701 calculates the ratio of the intensity value of the one known background peak on the peak list and the intensity value of the one known background peak on the exclusion list. The tandem mass spectrometer 701 then multiplies each intensity value on the peak list or the exclusion list by the ratio.

In various embodiments, two or more background ions can be used to scale the peak list or the exclusion list. For example, the tandem mass spectrometer 701 selects two or more known background peaks that are on the peak list and the exclusion list. The tandem mass spectrometer 701 calculates a ratio of the combination of the intensity values of the two or more known background peaks on the peak list and the combination of the intensity values of the two or more known background peaks on the exclusion list. The combination of the intensity values of the two or more known background peaks can be, without limitation, an average, median, or intensity-weighted average. The tandem mass spectrometer 701 then multiplies each intensity value on the peak list or the exclusion list by the ratio.

In various embodiments, the tandem mass spectrometer 701 further includes a sample introduction device 760. The sample introduction device 760 introduces, for example, one or more compounds of interest from a sample over time into the ion source device 710. The sample introduction device 760 can perform techniques including, but not limited to, injection, liquid chromatography, gas chromatography, capillary electrophoresis, or ion mobility.

When using the sample introduction device 760, the tandem mass spectrometer 701 receives an ion beam from the ion source device 710, which receives one or more compounds from the control sample over time from the sample introduction device 760. The tandem mass spectrometer 701 then performs multiple MS scans of a mass range at multiple different time steps on the ion beam to generate background peak m/z and intensity values for background precursor ions over time. At each time step, the tandem mass spectrometer 701 selects one or more of the background peaks of the background precursor ions for an exclusion list and includes the m/z value, intensity value, and residence time value for each background peak of the selected one or more of the background peaks in the exclusion list.

The tandem mass spectrometer 701 then receives an ion beam from the ion source device 710, which receives one or more compounds from the experimental sample over time from the sample introduction device 760. The tandem mass spectrometer 701 performs multiple MS scans of the mass range of the ion beam at multiple different time steps to generate peak m/z and intensity values for precursor ions over time. For each time step, the tandem mass spectrometer 701 selects one or more of the precursor ion peaks for a peak list and includes per-peak m/z values, intensity values, and dwell time values for the selected one or more peaks in the peak list.

Finally, the tandem mass spectrometer 701 excludes from the peak list each peak having an m/z value, intensity value, and retention time that corresponds to the m/z value, intensity value, and retention time of a background peak in the exclusion list.

In various embodiments, the m/z value, intensity value, and dwell time of a peak in the peak list corresponds to the m/z value, intensity value, and dwell time of a background peak in the exclusion list if the m/z value fits within the m/z tolerance factor, the intensity value fits within the intensity tolerance factor, and the dwell time value fits within the dwell time tolerance factor.

In various embodiments, a processor 750 can be used to control or instruct the tandem mass spectrometer 701 to perform any of the steps described above, or to independently perform one or more of the steps described above. The processor 750 controls or provides instructions, for example, by controlling one or more voltage, current, or pressure sources (not shown). The processor 750 can be, without limitation, a computer, a microprocessor, the computer system of FIG. 1, or any device capable of sending and receiving control signals and data to and from the tandem mass spectrometer, and processing data. The processor 750 communicates with the tandem mass spectrometer 701. Although the processor 750 is shown as a separate device, it can also be a processor or controller of the tandem mass spectrometer 701 or another device.
Method for excluding background peaks from the peak list

Figure 8 is a flow chart showing a method 800 for removing endogenous background ions with the same m/z as metabolite ions from a peak list in an IDA mass spectrometry experiment, according to various embodiments.

In step 810 of method 800, an ion beam produced by ionizing a control sample that does not contain metabolite compounds using a tandem mass spectrometer is received from an ion source device.

In step 820, a mass spectrometry (MS) scan of a mass range is performed on the ion beam using a tandem mass spectrometer to generate background peak m/z and intensity values for background precursor ions.

In step 830, using the tandem mass spectrometer, one or more of the background peaks of the background precursor ions are selected for an exclusion list, and in the exclusion list, m/z values and intensity values are included for each background peak of the selected one or more background peaks.

In step 840, an ion beam produced by ionizing an experimental sample containing metabolite compounds using a tandem mass spectrometer is received from an ion source device.

In step 850, an MS scan of the mass range is performed on the ion beam using a tandem mass spectrometer to generate peak m/z and intensity values for the precursor ions.

In step 860, using the tandem mass spectrometer, one or more of the precursor ion peaks are selected for a peak list, in which m/z values and intensity values are included for each of the selected one or more peaks.

In step 870, using a tandem mass spectrometer, each peak in the peak list that has both an m/z value and an intensity value that correspond to the m/z value and intensity value of a background peak in the exclusion list is excluded.

Computer program product for removing background peaks from a peak list

In various embodiments, a computer program product includes a tangible computer-readable storage medium, the contents of which include a program with instructions to be executed on a processor to perform a method for removing endogenous background ions having the same m/z as a metabolite ion from a peak list in an IDA mass spectrometry experiment. The method is performed by a system that includes one or more distinct software modules.

Figure 9 is a schematic diagram of a system 900 including one or more distinct software modules implementing a method for excluding endogenous background ions having the same m/z as metabolite ions from a peak list in an IDA mass spectrometry experiment, according to various embodiments. The system 900 includes a control module 910 and an analysis module 920.

The control module 910 instructs the tandem mass spectrometer to receive an ion beam from the ion source device that is generated by ionizing a control sample that does not contain metabolite compounds. The control module 910 instructs the tandem mass spectrometer to perform an MS scan of a mass range on the ion beam and generate background peak m/z and intensity values for the background precursor ions. The analysis module 920 selects one or more of the background peaks of the background precursor ions for an exclusion list and includes the m/z and intensity values for each background peak of the selected one or more background peaks in the exclusion list.

The control module 910 instructs the tandem mass spectrometer to receive an ion beam from an ion source device generated by ionizing an experimental sample containing metabolite compounds. The control module 910 instructs the tandem mass spectrometer to perform an MS scan of the ion beam's mass range and generate peak m/z and intensity values for the precursor ions. The analysis module 920 selects one or more of the precursor ion peaks for a peak list and includes in the peak list the per-peak m/z and intensity values of the selected one or more peaks. Finally, the analysis module 920 excludes from the peak list each peak having both an m/z value and an intensity value that correspond to the m/z value and intensity value of a background peak in the exclusion list.

Furthermore, in describing various embodiments, the specification may present the method and/or process as a particular sequence of steps. However, in that the method or process does not rely on the particular order of steps described herein, the method or process should not be limited to the particular sequence of steps described. As one of ordinary skill in the art would understand, other sequences of steps may be possible. Thus, the particular order of steps described herein should not be construed as a limitation on the claims. In addition, claims directed to methods and/or processes should not be limited to the performance of those steps in the order written, and one of ordinary skill in the art can readily understand that the sequence may be varied and still remain within the spirit and scope of the various embodiments.

Claims (15)

1. A system for removing endogenous background ions having the same mass/charge ratio (m/z) as a metabolite ion from a peak list in an Information Dependent Acquisition (IDA) mass spectrometry experiment, comprising:
an ion source device;
A tandem mass spectrometer, comprising:
(a) generating an exclusion list by receiving an ion beam from the ion source device generated by ionizing a control sample that does not contain metabolite compounds, performing a mass spectrometry (MS) scan of a mass range on the ion beam to generate background peak m/z and intensity values for background precursor ions, selecting one or more of the background peaks of the background precursor ions for an exclusion list, and including the m/z value and intensity value for each background peak of the selected one or more background peaks in the exclusion list;
(b) generating said peak list by receiving an ion beam from said ion source device generated by ionizing an experimental sample containing said metabolite compounds, performing an MS scan of said ion beam of said mass range to generate peak m/z and intensity values for precursor ions, selecting one or more of said precursor ion peaks for a peak list, and including per-peak m/z and intensity values of said selected one or more peaks in said peak list;
(c) excluding from the peak list each peak having both an m/z value and an intensity value that corresponds to an m/z value and an intensity value of a background peak in the exclusion list. The system of claim 1, wherein the m/z and intensity values of the peaks in the peak list correspond to the m/z and intensity values of the background peaks in the exclusion list if the m/z values fit within an m/z tolerance factor and the intensity values fit within an intensity tolerance factor. The system of claim 1, wherein the tandem mass spectrometer further scales the peak list or the exclusion list before comparing the peaks in step (c). The tandem mass spectrometer further comprises:
selecting a known background peak that is on the peak list and the exclusion list;
calculating a ratio of an intensity value of the one known background peak on the peak list and an intensity value of the one known background peak on the exclusion list;
4. The system of claim 3 , further comprising: scaling the peak list or the exclusion list by: multiplying each intensity value on the peak list or the exclusion list by the ratio. The tandem mass spectrometer further comprises:
selecting two or more known background peaks that are on the peak list and the exclusion list;
calculating a ratio of a combination of intensity values of the two or more known background peaks on the peak list and a combination of intensity values of the two or more known background peaks on the exclusion list;
4. The system of claim 3 , further comprising: scaling the peak list or the exclusion list by: multiplying each intensity value on the peak list or the exclusion list by the ratio. The tandem mass spectrometer further includes a sample introduction device, the tandem mass spectrometer further comprising:
In step (a), receiving an ion beam from the ion source device receiving one or more compounds from the control sample over time from the sample introduction device, performing a plurality of MS scans of the mass range on the ion beam at a plurality of different time steps to generate background peak m/z and intensity values for background precursor ions over time, and at each time step selecting one or more of the background peaks of the background precursor ions for the exclusion list, and including in the exclusion list the m/z value, intensity value, and residence time value for each background peak of the selected one or more of the background peaks;
In step (b), receiving an ion beam from the ion source device receiving one or more compounds from the experimental sample over time from the sample introduction device, performing a plurality of MS scans of the mass range on the ion beam at a plurality of different time steps to generate peak m/z and intensity values for precursor ions over time, and at each time step selecting one or more of the precursor ion peaks for the peak list, and including per-peak m/z values, intensity values, and dwell time values for the selected one or more peaks in the peak list;
In step (c), excluding from the peak list each peak having an m/z value, an intensity value, and a retention time value corresponding to an m/z value, an intensity value, and a retention time value of a background peak in the exclusion list;
The system of claim 1 . 2. The system of claim 1, wherein the m/z, intensity, and retention time values of a peak in the peak list correspond to the m/z, intensity, and retention time values of a background peak in the exclusion list if the m/z value fits within an m/z tolerance factor, the intensity value fits within an intensity tolerance factor, and the retention time value fits within a retention time tolerance factor. 1. A method for removing endogenous background ions having the same mass/charge ratio (m/z) as a metabolite ion from a peak list in an Information Dependent Acquisition (IDA) mass spectrometry experiment, comprising:
receiving an ion beam from an ion source device generated by ionizing a control sample that does not contain metabolite compounds using a tandem mass spectrometer;
performing a mass spectrometry (MS) scan of a mass range on the ion beam using the tandem mass spectrometer to generate background peak m/z and intensity values for background precursor ions;
using the tandem mass spectrometer to select one or more of the background peaks of the background precursor ions for an exclusion list and include an m/z value and an intensity value for each background peak of the selected one or more background peaks in the exclusion list;
using the tandem mass spectrometer to receive an ion beam from the ion source device that is generated by ionizing an experimental sample that includes the metabolite compounds;
performing an MS scan of the mass range on the ion beam using the tandem mass spectrometer to generate peak m/z and intensity values for precursor ions;
using the tandem mass spectrometer to select one or more of the precursor ion peaks for a peak list and include per-peak m/z and intensity values of the selected one or more peaks in the peak list;
and using the tandem mass spectrometer to exclude from the peak list each peak having both an m/z value and an intensity value that corresponds to an m/z value and an intensity value of a background peak in the exclusion list. The method of claim 8, wherein the m/z and intensity values of the peaks in the peak list correspond to the m/z and intensity values of the background peaks in the exclusion list if the m/z values fit within an m/z tolerance factor and if the intensity values fit within an intensity tolerance factor. The method of claim 8, further comprising scaling the peak list or the exclusion list before excluding from the peak list each peak having both an m/z value and an intensity value corresponding to an m/z value and an intensity value of a background peak in the exclusion list. Scaling the peak list or the exclusion list comprises:
selecting a known background peak that is on the peak list and the exclusion list;
calculating a ratio of an intensity value of the one known background peak on the peak list and an intensity value of the one known background peak on the exclusion list;
and multiplying each intensity value on the peak list or the exclusion list by the ratio. Scaling the peak list or the exclusion list comprises:
selecting two or more known background peaks that are on the peak list and the exclusion list;
calculating a ratio of a combination of intensity values of the two or more known background peaks on the peak list and a combination of intensity values of the two or more known background peaks on the exclusion list;
and multiplying each intensity value on the peak list or the exclusion list by the ratio. receiving an ion beam from the ion source device that receives one or more compounds from the control sample over time from a sample introduction device;
performing a plurality of MS scans of the mass range on the ion beam at a plurality of different time steps to generate background peak m/z and intensity values for background precursor ions over time;
At each time step, selecting one or more of the background peaks of the background precursor ions for the exclusion list and including in the exclusion list an m/z value, an intensity value, and a retention time value for each background peak of the selected one or more of the background peaks;
receiving an ion beam from the ion source device that receives one or more compounds from the experimental sample over time from the sample introduction device;
performing a plurality of MS scans of the mass range on the ion beam at a plurality of different time steps to generate peak m/z and intensity values for precursor ions over time;
At each time step, selecting one or more of the precursor ion peaks for the peak list and including per-peak m/z, intensity and retention time values of the selected one or more peaks in the peak list;
and excluding from the peak list each peak having an m/z value, an intensity value, and a retention time value that corresponds to an m/z value, an intensity value, and a retention time value of a background peak in the exclusion list. 9. The method of claim 8, wherein the m/z, intensity, and dwell time values of a peak in the peak list correspond to the m/z, intensity, and dwell time values of a background peak in the exclusion list if the m/z value fits within an m/z tolerance factor, if the intensity value fits within an intensity tolerance factor, and if the dwell time value fits within a dwell time tolerance factor . 1. A computer program product comprising a non-transitory, tangible computer readable storage medium, the contents of which include a program with instructions for execution on a processor to perform a method for removing endogenous background ions having the same mass/charge ratio (m/z) as metabolite ions from a peak list in an Information Dependent Acquisition (IDA) mass spectrometry experiment, the method comprising:
Providing a system, the system comprising one or more distinct software modules, the distinct software modules comprising a control module and an analysis module;
using the control module to instruct the tandem mass spectrometer to receive an ion beam from an ion source device that is generated by ionizing a control sample that does not contain metabolite compounds;
using the control module to instruct the tandem mass spectrometer to perform a mass spectrometry (MS) scan of a mass range on the ion beam to generate background peak m/z and intensity values for background precursor ions;
using the analysis module to select one or more of the background peaks of the background precursor ions for an exclusion list and include in the exclusion list an m/z value and an intensity value for each background peak of the selected one or more background peaks;
instructing the tandem mass spectrometer using the control module to receive an ion beam from the ion source device generated by ionizing an experimental sample containing the metabolite compounds;
using the control module to instruct the tandem mass spectrometer to perform an MS scan of the mass range on the ion beam to generate peak m/z and intensity values for precursor ions;
using the analysis module to select one or more of the precursor ion peaks for a peak list and include per-peak m/z and intensity values of the selected one or more peaks in the peak list;
and using the analysis module to exclude from the peak list each peak having both an m/z value and an intensity value that corresponds to an m/z value and an intensity value of a background peak in the exclusion list.

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